Oxindole–benzothiazole hybrids as CDK2 inhibitors and anticancer agents: design, synthesis and biological evaluation

In the current study, molecular hybridization between the oxindole core and benzothiazole system through an acetohydrazide moiety was accomplished for the design of a new series of oxindole–benzothiazole hybrids 9a–r targeting CDK2 for cancer therapy. The afforded hybrids displayed promising growth inhibitory activity on NCI cancer cell lines at 10 µM. Compound 9o displayed mean GI% = 55.91%. Based on the potent activity of 9o, it was further assessed for its cytotoxic activity at five dose level and it demonstrated GI50 reaching 2.02 µM. Analysis of the cell cycle of the prostate cancer cell line DU145 after treatment with 9o confirmed its ability to arrest its cell cycle at the G1 phase. Moreover, 9o proved its ability to potentiate the apoptosis and necrosis of the same cell line. Furthermore, the oxindole–benzothiazole hybrids 9b, 9f and 9o showed IC50 = 0.70, 0.20 and 0.21 µM, respectively on CDK2. Besides, molecular docking simulation of the synthesized oxindole–benzothiazole hybrid 9o proved the expected binding mode which involves the accommodation of the oxindole moiety in the ATP binding pocket where it is involved in hydrogen bonding and hydrophobic interactions with the essential amino acids in the hinge region while the benzothiazole moiety is oriented toward the solvent region. Investigation of the physicochemical properties of the hybrids 9a–r highlights their acceptable ADME properties that can be somewhat developed for the discovery of new anticancer agents. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-024-01277-1.


Introduction
Cancer is a global critical heterogeneous disease that arises as a result of unlimited proliferation of cells [1].Prescription of traditional chemotherapeutic agents was one of the main approaches for the treatment of cancer [2].However, it is always associated with unselectivity, severe side effects and toxicity.One approach to counteract this drawback is the prescription of a targeted therapy that targets a pathway that is overexpressed in cancer and plays a key role in controlling the proliferation of cancer cells without affecting the normal cells [3].One of these targeted therapies is the protein kinase inhibitors [3][4][5][6][7][8][9].
Cyclin-dependent kinases (CDKs) are a class of serine/ threonine kinases that participate directly in the regulation of the cell cycle besides their role in the regulation of growth, proliferation and apoptosis [10].CDK2 is a subtype from the CDK family that plays a major role in the mechanism of the cell cycle.Several studies reported the up-regulation of CDK2 in diverse types of cancer including breast cancer, prostate cancer, liver cancer and lung cancer [11].Hence, targeting CDK2 is considered a promising approach for controlling the progression of cancer [12].
On the other side, 2-aryl benzothiazole is a privileged scaffold that was reported in diverse molecules with promising anticancer activity and protein kinase inhibitory activity.CJM 126 (V) and NSC 703786 (VI) (Fig. 2) were reported to induce DNA damage in diverse cancer cell lines including breast, ovarian and colon cancer cell lines [25,26].Moreover, GW 610 (VII) (Fig. 2) revealed sub-nanomolar growth inhibitory activity in vitro against breast cancer [27].In addition, compound VIII (Fig. 2) was reported to exhibit high growth inhibitory potency of MCF-7 cell line [28,29].
Encouraged by the potent antiproliferative activity in conjunction with the privileged protein kinase inhibitory activity of both the oxindole and benzothiazole moieties we were curious in the current investigation to design a new scaffold of oxindole-benzothiazole hybrids IX and X (Fig. 3) as CDK2 inhibitors.The designed oxindole-benzothiazole scaffold IX and X was tailored so that the oxindole moiety was linked to 2-phenyl benzothiazole moiety Fig. 1 Examples of oxindole based protein kinase inhibitors I-IV through acetohydrazide linker.The oxindole moiety is expected to occupy the ATP binding site of CDK2 and perform hydrogen bonding with the key amino acid residues Glu81 and Leu83 through CONH group.The oxindole moiety is further settled in the ATP binding site by the ability of the fused benzene ring to form hydrophobic interactions with the side chains of the amino acids lining this region.The benzothiazole moiety is directed towards the solvent region.For studying the SAR, initially scaffold IXa (Fig. 3) was designed followed by the introduction of a methoxy group at the three position in IXb (Fig. 3) followed by regioisomersim of the oxindole moiety from the two position in scaffolds IXa and IXb (Fig. 3) to the three position in X (Fig. 3).The oxindolebenzothiazole scaffold was subsequently synthesized and submitted for screening their cytotoxic activity on different NCI cell lines derived from diverse types of cancer.The most potent hybrids were subsequently evaluated for their effect on the cell cycle and the apoptosis of a selected cell line.Additionally, the most potent candidate was docked into the binding site of CDK2 to confirm the design strategy.

Chemistry
The designed oxindole-benzothiazole hybrids 9a-r was synthesized according to the pathway depicted in Fig. 4 under reflux to afford the corresponding 2-substituted benzothiazole derivatives 3a-c [30,31].The hydroxy moiety of 3a-c was further functionalized by the basecatalyzed reaction of 3a-c with methyl bromoacetate (4) at room temperature to afford 5a-c which were further reacted with excess hydrazine hydrate (6) under reflux to yield the corresponding acid hydrazides 7a-c [30,32].
The benzothiazole acetohydrazides 7a-c were further reacted with diverse oxindoles 8a-f under acidic conditions to afford the target derivatives 9a-r in good yields (Fig. 4).The structures of the afforded derivatives were further confirmed by IR, 1 H NMR and 13 C NMR spectra (for further details see Additional file 1: NMR Spectra of oxindole-benzothiazole hybrids 9a-r; IR charts of the synthesized oxindole-benzothiazoles).For instance, the IR spectrum of 9a showed the appearance of two bands at ṽ 3221 and 3148 cm −1 corresponding to NH groups; two bands at ṽ 3059 and 3036 cm −1 corresponding to aromatic CH; a band at ṽ 2959 cm −1 corresponding to aliphatic CH; two bands at ṽ 1721 and 1694 cm −1 corresponding to CO.

Screening of the antiproliferative activity on NCI cancer cell lines at single dose concentration
The oxindole-benzothiazole conjugates 9a-c and 9e-r were assayed for their potential to inhibit the growth of cancer cell lines that originate from diverse types of cancer after treatment with 10 µM concentrations at NCI-USA and the results were depicted in Table 1 and compared with milciclib as a standard (Additional file 1: screening of cytotoxic activity against a panel of sixty human tumor cell lines; one dose mean graphs of the oxindole-benzothiazoles).
The oxindole-benzothiazole hybrids 9a-r displayed disparate growth inhibitory activity on NCI cell lines.The synthesized derivatives demonstrated mean growth inhibition percentage spanning from < 5% to 55.91% in reference to milciclib which showed a mean growth inhibitory activity more than 100% (Table 1).

Antiproliferative activity of 9o on NCI cancer cell lines at five concentrations
Encouraged by the potent activity of 9o on diverse cancer cell lines on the one-dose assay (Table 1), it was further selected to be examined at 5-dose concentrations and the GI 50 was depicted in Table 2 and Fig. 6 (for additional details see Additional file 1: dose-response curves of 9o on NCI cancer cell lines).The oxindole-benzothiazole hybrid 9o revealed moderate to potent potency against the tested cell lines (GI 50 reaching 2.02 µM).Close examination showed that 9o displayed GI 50 of 3.75 µM on the K-562 cell line from leukemia, GI 50 = 3.03 µM on the NCI-H23 cell line from non-small cell lung cancer.HCT-116, HCT-15 and SW-620 cell lines from colon cancer are sensitive to 9o with GI 50 = 4.50, 3.60 and 2.27 µM, respectively.Also, the U251 cell line from CNS cancer is very sensitive to 9o (GI 50 = 2.02 µM).Additionally, 9o demonstrated GI 50 = 4.09 and 2.28 µM on LOX IMVI and MALME-3M cell lines, respectively from melanoma; GI 50 = 2.22, 2.49 and 4.02 µM on IGROV1, OVCAR-3 and OVCAR-8 cell lines, respectively from ovarian cancer;

Effect of 9o on the cell cycle of DU145 prostate cancer
Motivated by the potent activity of 9o on prostate cancer cell lines in Table 2, it was further examined for its effect on the cell cycle of the DU145 cell line at its GI 50 concentration and the results were depicted in Fig. 7 and Table 3. Obviously, 9o proved the ability to arrest the cell cycle of the DU-145 cell line at the G1 phase as the % of cells accumulated in the G1 phase raised from 57.91% in control cells to 61.40% in 9o treated cells.Concurrently, there is a decline in the % of cells in the G2 phase from 22.20% in control cells to 20.94% in 9o treated cells.

Apoptotic effect of 9o on DU145 prostate cancer
In parallel, the capability of 9o to potentiate the apoptosis of the DU145 cell line was explored at its GI 50 concentration.The presented results in Fig. 8 confirm the potency of 9o to induce the apoptosis and necrosis of the DU145 cell line as the % of cells in the late apoptotic stage elevated from 2.27% in control cells to 5.02% in treated cells.Also, Fig. 8, showed that 9o increased the number of cells in the necrotic stage from 0.67% in control cells to 2.63% in treated cells.

Inhibitory activity of selected candidates on CDK2
The oxindole-benzothiazole conjugates 9b, 9f and 9o were assayed for their potential to suppress the activity of CDK2 and the results were represented as the IC 50 in µM and compared with staurosporine as a standard (Table 4).
From the obtained results it is obvious that compounds 9b, 9f and 9o are potential inhibitors of CDK2 with IC 50 = 0.70, 0.20 and 0.21 µM.Compounds 9f and 9o revealed the most potent inhibitors followed by 9b (Table 4).

Inhibitory activity of 9o on diverse kinases
Subsequently, the conjugate 9o was examined for its inhibitory activity on CDK1 and CDK5 isoforms as well as for its inhibitory activity on VEGFR-2 and FGFR-1 and the outcomes were presented in Table 5.

Molecular docking simulation
To confirm the expected mode of binding of the oxindole-benzothiazole hybrids 9a-r to CDK2, compound 9o was selected to be docked into the binding pocket of CDK2 using Autodock Vina [33] and the results were visualized using BIOVIA Discovery Studio Visualizer https:// disco ver.3ds.com/ disco very-studio-visua lizer.
First, the crystal structure of CDK2 (PDB ID: 1FVT) [34] was retrieved from the protein data bank and the protein was prepared followed by re-docking of the native ligand to validate the protocol that will be employed for the docking study (for further details see Additional file 1: docking of the co-crystalized ligand in the binding site of CDK2).Afterward, the oxindole-benzothiazole hybrid 9o was docked into CDK2's binding pocket and the results were analysed [16].The synthesized oxindole-benzothiazole hybrid 9o expressed higher affinity to the active site of CDK2 with docking energy scores (S) − 10.8 kcal/mol in relevance to the native ligand docking energy score (S) of − 9.1 kcal/mol.As shown in Fig. 9, the oxindole part of the oxindole-benzothiazole scaffold 9o is settled in the ATP binding pocket where the lactam ring performs hydrogen bonding with the key amino acids Glu81 and Leu83, and the NH group of the acetohydrazide is involved in hydrogen bonding with Leu83, while the fused phenyl ring participates in hydrophobic interactions with the adjacent amino acid residues Val18, Ala31, Leu134, Ala144 and Asp145.Meanwhile, the 2-phenyl benzothiazole moiety is directed toward the solvent region where it creates hydrophobic interactions with the amino acids Ile10, Lys20, Lys89, Arg297 and Leu298 at the binding pocket's entrance (Fig. 9).

ADME properties prediction
The synthesized oxindole-benzothiazole hybrids 9a-r were tested using the SwissADME online tool to determine their drug similarity and ADME characteristics [35].Table 6 demonstrates some selected findings.The majority of the hybrids 9a-r satisfy Lipinski's criterion of 5 [36][37][38], the derivatives 9f, 9j, 9l, 9p and 9r are the only instances that exhibit one or two violations.It is anticipated that none of the submitted oxindole-benzothiazole hybrids 9a-r are sufficiently lipophilic to cross the blood-brain barrier, highlighting the absence of any anticipated central effects [39].All the synthesized candidates are not substrates to P-glycoprotein (P-gp) which is the primary transporter of xenobiotics to the outside of the cells [40].The majority of the provided hits have a bioavailability score of 0.55, indicating that they are mostly orally bioavailable.Furthermore, the bioavailability radar charts of oxindole-benzothiazoles 9b, 9f and 9o are shown in Fig. 10 (for further details see additional file 1: bioavailability radar charts for 9a-r from Swis-sADME free webtool).They highlight ideal size, polarity, flexibility, and solubility for oral bioavailability.The only characteristic that deviates slightly from its ideal value is the degree of saturation.As a conclusion, we can summarize that in addition to the potential CDK2 inhibitory action as targeted anticancer agents, the oxindole-benzothiazole hybrids 9a-r displayed acceptable ADME qualities that can be further optimized as anticancer agents.

Conclusion
The construction of a new scaffold of oxindole-benzothiazole conjugates 9a-r as CDK2 inhibitors and anticancer drugs was accomplished through the use of the molecular hybridization technique.The scaffold was synthesized using conventional organic synthesis techniques.Various spectral data were utilized to verify the structures of the afforded candidates.Examining the produced candidates' growth inhibitory activity on NCI cancer cell lines demonstrated their weak to strong growth inhibitory effect.Specifically, 9o displayed a strong GI 50 that reached 2.02 µM.DU145 cell line from prostate cancer was examined for how 9o affected its cell cycle, and it was found that 9o stopped the cell cycle at the G1 phase.Additionally, 9o demonstrated its capacity to induce late apoptosis and necrosis, which accelerate the cell death of the DU145 cell line.Additionally, the oxindole-benzothiazole conjugates 9b, 9f and 9o showed potent CDK2 inhibitory activity with IC 50 = 0.70, 0.20 and 0.21 µM, respectively.Moreover, 9o was found to have higher selectivity toward CDK2 and CDK5 over CDK1, VEGFR-2 and FGFR-1.In silico docking of 9o into CDK2 active site proved the predicted binding mode in which the oxindole moiety is settled in the ATP binding pocket and is involved in hydrogen bonding interactions with the key amino acids Glu81 and Leu83 as well as hydrophobic interaction with the amino acid residues lining the hinge region, while the benzothiazole moiety is directed towards the solvent region.Additionally, the proposed oxindole-benzothiazole hybrids 9a-r exhibit acceptable physicochemical and pharmacokinetics qualities that can be further optimized as anticancer agents.

General remarks
Chemicals that were used in organic synthesis and for biological screening were picked up from commercial companies.The chemical reactions were followed up employing pre-coated silica gel 60 F 245 aluminium plates (Merck).Melting points of the synthesized molecules were recorded on a Stuart SMP30 melting point instrument.Spectroscopic measurements and elemental analysis of the synthesized organic derivatives were afforded in the Micro analytical labs, National Research Centre, Cairo, Egypt.A Jasco FT/IR 300 E Fig. 7 Cell cycle of DU145 before and after treatment with 9o  Fourier transform infrared spectrophotometer was used for measuring the IR spectra (4000-400 cm −1 ).Bruker instruments 500 (125) MHz and 400 (100) MHz were used for recording the 1 H NMR and 13 C NMR (DMSO-d 6 ).

Fig. 5
Fig. 5 Structure-activity relationship of 9a-c and 9e-r on NCI cancer cell lines

Table 1
In vitro growth inhibition% (GI%) of NCI 60 cancer cell line panel after treatment with 10 μM of the oxindole-benzothiazoles 9a-

Table 1 (
continued) b Not detected

Table 3
Different phases of cell cycle of DU145 before and after treatment with 9o

Table 4
Inhibitory activity of the oxindole-benzothiazole conjugates 9b, 9f and 9o on CDK2 a Results are mean of two independent experiments ± standard deviation (SD)